Decellularisation of tissue matrices for bladder implantation

ABSTRACT

The invention provides an improved method of producing a natural, acellular matrix scaffold for subsequent use in tissue-engineered replacement of tissues such as the bladder. Decellularization is carried out on an expanded or distended bladder and the product retains the strength and compliance of natural material. The invention also provides use of the matrix scaffolds as wound healing material and to investigate tissue structure and function in vitro.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/295,190, now allowed, filed on Sep. 29, 2008, which claims priorityto and is a 35 U.S.C. §371 national phase application of PCTInternational Application No. PCT/GB2007/001117, having an internationalfiling date of Mar. 28, 2007 and claiming priority to Great BritainPatent Application No. 0606231.9, filed Mar. 29, 2006. The disclosuresof each application are incorporated herein by reference in theirentireties. The above PCT International Application was published in theEnglish language and has International Publication No. WO 2007/110634A2.

The present invention relates to a method of producing a natural,acellular matrix scaffold for subsequent use in tissue-engineeredreplacement of tissue. The invention provides a biomaterial that issuitable for tissue replacement and/or repair particularly but notexclusively in the fields of gastroenterology, urology, cosmeticsurgery, wound repair and reconstructive surgery. The invention alsoprovides use of the matrix scaffolds to investigate tissue structure andfunction in vitro.

BACKGROUND

At present, natural biomaterials are predominantly used inreconstructive surgery, mainly in the fields of gastroenterology,urology and wound-healing, but with increasing applications incardiovascular and cosmetic surgery. One such biomaterial, porcine smallintestinal submucosa (SIS), has been used in gastroenterology, urologyand wound-healing applications as it is easily incorporated into hosttissue and remodeled. However, recent work to investigate the adherenceand viability of human cells seeded onto SIS demonstrated thatcommercially-available SIS specimens contained porcine nuclear residuesand was cytotoxic in vitro. In addition, clinical use of SIS hasresulted in localised inflammation, suggesting the material can cause animmunological response in vivo. An alternative natural biomaterial isdecellularised porcine dermis (Permacol™) which has a wide range of usesin medical and cosmetic procedures and has been implanted in over 8500patients in over 70 different surgical procedures since being licensedfor use in humans in 1998. However, Permacol™ a disadvantage associatedwith this material is that not only is it only partially resorbable butwhen it is used in an animal model of bladder augmentation it has beenshown to cause micro-calcification and irregular detrusor regeneration.Furthermore, Permacol™ is unable to support in vitro recellularisationthus preventing its use as a biomaterial that could be seeded with cellsand functionalised prior to implantation. An improved naturalbiomaterial that is immunologically inert and able to supportrecellularisation would offer an immediate advantage in the art.

It is estimated that over 400 million people worldwide suffer from someform of bladder dysfunction. A variety of diverse congenital andacquired conditions result in bladder dysfunction, for example cancer,congenital abnormalities, nerve damage or trauma. Currently, the majorsurgical solution is surgical reconstruction. It is known in the priorart to repair or augment or replace the bladder during these procedureswith vascularised segments of the patient's own tissue derived fromtheir stomach or more commonly their intestine. However, this latterprocedure (‘enterocystoplasty’) is associated with significant clinicalcomplications that arise due to the exposure of the epithelial lining ofthe intestine to urine. It has been found that the use of intestineresults in significant complications, such as infection and developmentof bladder stones, as the intestine is lined by an absorptive andmucus-secreting epithelium that is incompatible with long-term exposureto urine. Consequently, a number of alternative approaches have beenproposed to find a practical and functional substitute for nativebladder tissue. One of the alternative solutions is ‘compositeenterocystoplasty’, where the de-epithelialized intestine wall is linedwith bladder epithelial cells that have been propagated in vitro, toaugmenting the urinary system with natural or synthetic biomaterialsthat may incorporate in vitro-propagated cells. However even thismodified form of enterocystoplasty has been associated with adverse sideeffects. The lack of an entirely satisfactory clinical procedure has ledresearchers to investigate alternative strategies.

Attempts have been made to develop suitable biomaterials for urologicaltissue engineering using both synthetic materials, for examplepolyglycolic acid and poly L-lactic acid, and naturally derivedmaterials including SIS, Permacol™ and porcine bladder matrix. Howevernone of these materials have been found to be totally successful vis avis immunogenicity and rejection and recellularisation.

Tissue matrices prior to implantation undergo a process ofdecellularisation in order to reduce their immunogenicity onceimplanted. This process involves removing the donor cells, whilstideally retaining the biomechanical structure and function of thematrix. As regards the bladder, it is especially desirable to maintainthe normal mechanical properties and its elasticity.

A problem associated with the use of a bladder matrix is that bladdertissue is relatively thick (1-5 mm when not distended). This in turnmeans that it is difficult to decellularise bladder tissue to provide animmunologically inert scaffold matrix using routine methods known in theart. Attempts to decellularise dissected segments of full thicknessporcine bladder have resulted in incomplete decellularisation.Histological analysis of these samples indicated that cells had not beenremoved from the muscular bladder wall. In its retrieved form, theporcine bladder was too thick to allow efficient penetration of thesolutions used throughout the decellularisation process. In order toovercome this problem segments of bladder tissue of reduced thicknesscould be used to enable successful decellularisation.

An improved method of decellularisation that could be used todecellularise whole bladders or other membranous sacs of full thickness,whilst retaining the biomechanical properties of the tissue, would offerimmediate advantage in the field of particularly but not exclusively,urological tissue engineering.

BRIEF SUMMARY OF THE DISCLOSURE

According to a first aspect of the invention there is provided a methodof decellularisation of a tissue comprising a distensible membranoussac, the method comprising:

-   -   (i) immersing the distensible membranous sac in a buffer        solution at a mild alkaline pH which includes active amounts of        a proteolytic inhibitor;    -   (ii) distending the distensible membranous sac by introducing a        sufficient volume of the same buffer solution into the interior        cavity of the sac and;    -   (iii) continuing decellularisation of the sac by replacing and        introducing fresh solutions both around the exterior surface of        the sac and into the sac interior itself so as to maintain        distension of the sac during decellularisation.

It will be appreciated that step (ii) may be performed prior to step (i)and that the essence of these two steps is to ensure that both theinside and outside of the distensible membranous sac is in contact withthe buffer for effective decellularisation to take place. Whether thesac is fluid filled with the buffer and then immersed in the buffer orwhether it is immersed in the buffer and then filled with the buffer isnot material to the method providing that the sac is in contact with thebuffer on both its inner and outer membranous surfaces and that itremains in a distended form whilst decellularisation occurs.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, means “including but not limited to”, andis not intended to (and does not) exclude other moieties, additives,components, integers or steps.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

Reference herein to a distensible membranous sac is intended to includea tissue or sac or balloon of tissue that has an elastic or flexiblemembrane. That is to say tissue when in vivo has the ability to expandand to return to a normal resting state without rupture or disruption ofits mechanical properties.

Reference herein to decellularisation is intended to include the removalof cellular membranes, nucleic acids, lipids, cytosolic components andretaining an ECM having as major components collagens and elastins.

Preferably, the distensible membranous sac is clamped before and aftereach step of fluid replacement so as to prevent fluid loss and tomaintain distension of the sac.

Preferably, the fluid volume added to the interior cavity of the sac ateach step is in the region of 250-750 ml and more preferably in theregion of 350-650 ml and more preferably still is about 500 ml. Thevolume selected is dependent on the size of the distensible sac and mayvary accordingly, the volume introduced into the sac is sufficient tomaintain distension throughout the decellularisation process.

Preferably, the fluid volume around the exterior of the sac issufficient to cover the whole of the distended fluid filled sac. It isconvenient whilst refreshing the fluid inside the sac to simultaneouslyreplace the fluid surrounding the exterior of the sac.

Once the process of decellularisation is complete the distensible sacmay be dissected to form a flattened sheet or it may remain intact as asac depending on its intended subsequent use.

The bladder is an extremely compliant organ and is able to expand tomore than 15 times its contracted volume, it this property that has beenexploited in the method of the present invention. By distending theintact bladder to stretch and thin the bladder wall, solutions were ableto permeate throughout the thinned wall, resulting in completedecellularisation whilst surprisingly retaining properties of nativebladder tissue.

Preferably, the distensible membranous sac is a whole bladder.

Preferably, the distensible membranous sac is derived from a pig.

However, it will be appreciated that the method of the present inventionis equally applicable a membranous sac derived from a human or any otheranimal that has physical parameters, for example size, that arecompatible with a human bladder. Thus the method of the invention may beused to decellularise donor human bladders. It may also be used to, forexample, replace the bladder from another animal such as a cat with amembranous sac derived from a cat or another species which hasapproximately the same size as a cat's bladder. The interspecies crossis not intended to limit the scope of the invention.

Porcine whole bladder is the particularly preferred tissue for use inthe method of the present invention since not only is there a readysupply but its size, physical and mechanical characteristics are similarto humans which makes it compatible as a human bladder tissuereplacement.

Preferably, the method of decellularisation of a tissue comprising adistensible membranous sac, comprises:

-   -   (i) distending the distensible membranous sac by introducing a        sufficient volume of a buffer solution at a mild alkaline pH        which includes active amounts of a proteolytic inhibitor into        the sac whilst simultaneously immersing the sac in the same        buffer solution;    -   (ii) removing said buffer from both the interior cavity of the        sac and the surrounding exterior area and replacing it with an        anionic detergent at a mild alkaline pH at a concentration which        is sufficient to effect decellularisation but which maintains        the histoarchitecture of the biological material;    -   (iii) removing said detergent from the interior of the sac and        its exterior and replacing it with a washing buffer solution at        a mild alkaline pH both with and without active amounts of        proteolytic inhibitors so as to wash both the interior and        exterior surfaces of the distensible membranous sac;    -   (iv) removing said washing buffer from both the interior of the        sac and its exterior and replacing it with a solution comprising        one or more enzymes selected from the group comprising DNase        Type I, DNase Type II, and/or Rnase and optionally;    -   (v) removing the solution comprising one or more enzymes from        the interior of the sac and its exterior and optionally placing        the biological material in a cryoprotectant medium or storage        medium or other suitable protective medium for later use.

Preferably, the method further includes the step of modifying thedecellularised matrix with a suitable agent to enhanceimmunoacceptability of the matrix on implantation. For example andwithout limitation, the tissue matrix may be treated enzymatically withα-galactosidase or a glycosidase digestion to remove α-gal epitopes (Galα1-3 Gal β1-4Glc NAC-R). Alternatively, it may be treated chemicallywith cross-linking agents such as glutaraldehyde or other aliphatic andaromatic diamine compounds that provide additional cross-linking throughside chain groups of aspartic and glutamic acid residues of the collagenpeptide. It will be appreciated that any suitable agent that is capableof enhancing immunoacceptability and thus reducing the likelihood ofpost implantation rejection and/or inflammation will be applicable foruse in the method of the present invention.

Preferably, the method further includes the step of recellularisation.Recellularisation can be either in vitro or in vivo and may be enhancedby a suitable agent appropriately administered either in vitro or invivo. The agent may also be coated directly onto the tissue matrix priorto implantation to encourage recellularisation.

As regards the solutions and concentrations of components in the methodof the invention, preferably the buffer solution is hypotonic orisotonic. It will be appreciated that each may be used either as thesole buffer or in combination at different stages of the method and thatuse of hypotonic or isotonic buffer is not intended to limit the scopeof the present application.

Preferably, the proteolytic inhibitors are ethylene diamine tetraaceticacid (EDTA) and Aprotinin. We have found Aprotinin particularlyeffective as a proteolytic inhibitor and of particular utility becauseof its low toxicity, stability in solution at different pHs andstability at a variety of different temperatures.

Typically, EDTA is used at a concentration range of 1 to 100 mM or0.01-1.0% (w/v) and typically at 10 mM or 0.1% and Aprotinin at aconcentration range of 1-100 KIU and typically at 10 KIU.

Preferably, the mild alkaline conditions of step (i) are in the range ofpH above 7.0 and up to pH 10.0, and more preferably are at pH 8.0.

Preferably, the incubation period of step (i) of the method is forbetween 8 to 24 hours and more preferably is for 14 hours.

Preferably, the anionic detergent is sodium dodecyl sulphate (SDS) orsodium deoxycholate.

Preferably, SDS is used at a concentration equal to or below 0.1% (w/v),and equal to or above 0.03% (w/v).

Reference herein to the term % (w/v) refers to the percentage in weight(grams) per unit volume (100 ml), thus 0.1% w/v is equivalent to 0.1 gmdissolved in 100 ml.

Preferably, sodium deoxycholate is used at a concentration equal to orbelow 2.0% (w/v) and equal to or above 0.5% (w/v).

Preferably, the incubation period of step (ii) of the method is forbetween 20 to 28 hours and more preferably is for 24 hours.

Preferably, the mild alkaline conditions of step (ii) are in the rangeof pH above 7.0 and up to pH 10.0, and more preferably are at pH 8.0.

Preferably, the washing step (iii) of the method involves multiplewashes, typically ×3 washes with physiological buffered saline(preferably phosphate buffered saline PBS, 0.01M phosphate buffer,0.137M NaCl) containing protease inhibitors (0.1% EDTA and 10 KIU/mlAprotinin), and further, multiple washes, typically ×3 washes withphysiological buffered saline without the protease inhibitors.

Preferably, the mild alkaline conditions of step (iii) are in the rangeof pH above 7.0 and up to pH 10.0, and more preferably are at pH 8.0.

Preferably, the incubation step (iv) of the method is for 4-6 hours attemperature range of between 20° C. to 45° C. and preferably at 37° C.

The DNase Type I, DNase Type II or Rnase are employed in an amounteffective so as to eliminate nucleic acids and provide a tissue matrixof limited calcification potential. Accordingly any other agents whichare capable of the same function are included within the scope of thepresent invention.

Preferably, DNAse I is used at a concentration range of 5.0-100 μg/mland typically at 20 μg/ml and RNAse A at a concentration range of 0.1-10μg/ml and typically at 1 μg/ml.

The decellularisation process includes osmotic lysis of the cells,solubilisation of cell fragments using SDS, protease inhibitors toinhibit autolysis and nucleases to digest nuclear materials. All stagesof the process are carried out using a whole, full thickness distensiblesac that is substantially distended during all steps so that its wallsare stretched and thinned so allow solutions to permeate throughout thethinned wall, resulting in complete decellularisation thereof.

The method of the present invention provides a process whereby intactwhole porcine bladders are distended in a series of decellularisationsolutions such that decellularisation of the full thickness bladder wallis achieved. Moreover, the resultant biomaterial retained the physicaland structural features of the native bladder tissue and contained noresidual cell or nuclear bodies. Results have shown that the underlyingbladder histoarchitecture was retained and that the biomaterial producedby the method of the invention when compared to fresh porcine bladdertissue showed no differences in the overall compliance; ultimate tensilestrength or ability of the decellularised matrix to retain sutures underforce. Furthermore, the bladder biomaterial was biocompatible withcells, as homologous smooth muscle cells were able to repopulate thematrix after 21 days in culture.

Our results have shown there was no significant difference in theultimate tensile strength of the distensible sac followingdecellularisation and in terms of its potential applications in vivo,there was no significant difference in the ability of the decellularisedmatrix to retain sutures under force compared to fresh bladder tissue.

According to a further aspect of the invention there is provided anatural biomaterial product comprising bladder tissue having a DNAcontent of less than 0.2 μg/mg dry weight, a suture retention strength(Fmax) of between 3-6 N, ultimate tensile strength (apex to base) ofbetween 1-4 MPa, failure strength (apex to base) of between 70-150%.

Preferably, the natural biomaterial product is a whole bladder or is apatch or portion of bladder tissue. It will be appreciated that thenatural biomaterial produced by the method of the present inventionsretains the above characteristics and parameters and as such may be usedeither as the whole bladder for transplantation or experimental purposesalternatively patches or portions of the bladder may be used for any ofthe various surgical uses and scenarios as hereinbefore mentioned.

The biocompatible implant material produced by the method of the presentinvention is characterised by a DNA content of less than 0.2 μg/mg dryweight, that is to say it is substantially decellularised. It is alsocharacterised by a suture retention strength (Fmax) of between 3-6 N,ultimate tensile strength (apex to base) of between 1-4 MPa, failurestrength (apex to base) of between 70-150% all of these parametersindicate that the mechanical strength of the biomaterial product iscomparable to natural bladder that has not undergone a decellularisationprocess according to the method of the invention.

The biomaterial of the present invention is stable, non-cytotoxic andretains the strength and compliance properties of the native bladder,indicating potential use in a range of surgical procedures and as ascaffold for bladder tissue-engineering.

According to a yet further aspect of the invention there is provided atissue matrix obtainable by the method of the present invention for useas a transplant tissue.

According to a yet further aspect of the invention there is provided useof a tissue matrix obtainable by the method of the present invention asa transplant tissue.

Preferably, the tissue matrix is a bladder tissue matrix.

Preferably, the bladder tissue matrix is used for reconstructive surgeryand in particularly in reconstruction surgery of congenital or acquiredbladder defects or bladder augmentation.

Initial studies in vitro using alternative biomaterials have provedencouraging in so far as normal urothelial cells (NUC) have been shownto readily attached and grow as a monolayer on the surface of Permacol™,however smooth muscle (SM) cells could only survive when co-culturedwith NUC (Kimuli et al BJU Int. 94, 859, 2004) and even when co-culturedwith NUC cells the SM cells could not infiltrate the Permacol™. Theseobservations confirm similar finding with SIS matrices and NUC and SMcells (Zhang et al J Urol, 928, 164, 2000). A functionally normalurinary bladder distends passively as it fills with urine, and contractsvoluntarily to void. To carry out this normal function the bladderrequires a normal SM cell component. We have found that the tissuematrices produced by the present invention could sustain SM cellproliferation, thus making it a suitable biomaterial for urologicaltissue engineering. Thus the improved method of decellularisationprovided in the present invention shows that distensible sacs treatedwith the method can, when implanted sustain two of the essential celltypes vital for normal function.

According to a yet further aspect of the invention there is provided atissue matrix obtainable by the method of the present invention for usein tissue repair.

According to a yet further aspect of the invention there is provided useof a of a tissue matrix obtainable by the method of the presentinvention in tissue repair.

It is envisaged that the processed bladder biomaterial will have uses intissue repair of for example and without limitation as pubovaginalslings and in vaginal repair. It is also envisaged that the bladderbiomaterial obtainable by the method of the present invention and havingthe unique characteristics as hereinbefore described has potentialapplications in gastroenterology including the repair of abdominal walldefects, such as those caused by trauma, tumour resection, and also inchest wall repair as pericardial patches following tumour excision andcould potentially be used for cardiac repair.

According to a yet further aspect of the invention there is provided atissue matrix obtainable by the method of the present invention for useas in wound healing or wound repair.

According to a yet further aspect of the invention there is provided useof a of a tissue matrix obtainable by the method of the presentinvention as wound healing or repair material.

Preferably, the processed bladder biomaterial is in the form of a sheetor patch.

In wound care, natural biomaterials can be used in the repair of defectsoccurring as a result of burns, venous ulcers, diabetic ulcers and largefull-thickness defects such as those occurring following acute injury.

According to a, yet further aspect of the invention there is provided atissue matrix obtainable by the method of the present invention for useas in cosmetic surgery as augmentation material

According to a yet further aspect of the invention there is provided useof a of a tissue matrix obtainable by the method of the presentinvention in cosmetic surgery as augmentation material.

Cosmetic applications for the processed bladder biomaterial includeeyebrow augmentation, repair of nasal septum, cleft palate repair,breast surgery, lip, cheek and chin augmentation.

The compliance the biomaterial we have developed also makes it asuitable material for soft and connective tissue coverage, whereintimate contact between the matrix and wound surface is required. Acustom made patch could be fashioned to fit to the margins of ulcers orwounds with the confidence that it would lie against deep structures inorder to protect and encourage healing. This may be of particularbenefit in traumatic tissue loss where myocutaneous flaps are currentlyused to cover exposed bony or tendonous structures. The ability of ourbiomaterial to conform to underlying structures would make it ideal foraugmentation cystoplasty, urothelial coverage in detrusor myectomy andother urological reconstructive procedures.

According to a yet further aspect of the invention there is provided atissue matrix obtainable by the method of the present invention for useas a scaffold for tissue-engineering.

According to a yet further aspect of the invention there is provided useof a tissue matrix obtainable by the method of the present invention asa scaffold for tissue-engineering.

According to a yet further aspect of the invention there is provided atissue matrix obtainable by the method of the present invention for useas an in vitro model to investigate bladder function.

According to a yet further aspect of the invention there is provided useof a tissue matrix produced by the method of the present invention as anin vitro model to investigate bladder function.

According to a yet further aspect of the invention there is provided amethod of implantation of a tissue matrix in bladder reconstruction orreplacement surgery comprising:

-   -   (i) immersing the distensible membranous sac in a buffer        solution at a mild alkaline pH which includes active amounts of        a proteolytic inhibitor;    -   (ii) distending the distensible membranous sac by introducing a        sufficient volume of the same buffer solution into the interior        cavity of the sac and;    -   (iii) continuing decellularisation of the sac by replacing and        introducing fresh solutions both around the exterior surface of        the sac and into the sac interior itself so as to maintain        distension of the sac during recellularisation and;    -   (iv) implanting either the whole decellularised distensible sac        or a portion thereof into a donor

As previously mentioned steps (i) and (ii) may be reversed.

According to a yet further aspect of the invention there is provided amethod of wound healing and/or tissue repair comprising:

-   -   (i) immersing the distensible membranous sac in a buffer        solution at a mild alkaline pH which includes active amounts of        a proteolytic inhibitor;    -   (ii) distending the distensible membranous sac by introducing a        sufficient volume of the same buffer solution into the interior        cavity of the sac;    -   (iii) continuing decellularisation of the sac by replacing and        introducing fresh solutions both around the exterior surface of        the sac and into the sac interior itself so as to maintain        distension of the sac during recellularisation and;    -   (iv) placing a portion or patch of the decellularised sac onto        or around a wound or attaching said portion or patch on or about        the tissue to be repaired.

Preferably, any of the further aspects of the invention further includeany one or more of the features recited in the first aspect of theinvention.

The method of the present invention, products produced thereby and theuse of the products provide a decellularised, biocompatible bladdermatrix which retains properties of native bladder tissue.

It is envisaged that the acellular matrix retaining the major structuralcomponents and strength of the urinary bladder can be used to produce afunctional, tissue-engineered construct for use in bladder repair and asan in vitro model to study host cell-matrix interactions and the role ofmechanical forces on bladder tissue functionality.

Surgeons who have felt the bladder biomaterial obtainable by the methodof the present invention have reported that the strength, feel,compliance and elasticity of the material makes it particularly wellsuited for any surgical procedures where Permacol™ and other naturalbiomaterials are being used currently. Indeed it has been indicated thatthe compliance of the material of the present invention makes it a moreattractive material to use than available prior art materials.

Furthermore experimental evidence suggests that decellularised bladdermaterial has a good shelf life even after as long as 30 months storage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a photomicrograph of dissected bladder showing the fiveanatomical regions of the bladder that underwent biomechanicalcharacterisation. Tissue strips from all regions were collected in bothtransverse (T) and base-to-apex (B-A) directions.

FIG. 2 shows histological characterisation of fresh (A, B) anddecellularised (C, D) porcine bladder. Sections were stained withhaematoxylin and eosin (A and C) and Hoeschst 33258 (B and D). Scalebar—100 μm.

FIG. 3 shows histological characterisation of fresh (A and C) anddecellularised (B and D) porcine bladder. Paraffin-wax-embedded sectionswere stained with Van Gieson (A and B) and Miller's Elastin stain (C andD) [SM: Smooth Muscle, C: Collagen matrix]. Scale bar—100 μm.

FIG. 4 shows immunoperoxidase labelling of fresh (A, C, E and G) anddecellularised (B, D, F and H) porcine bladder. Sections labelled withantibodies against collagen type I (A and B), smooth muscle actin (C andD), vimentin (E and F) and desmin (G and H) were counterstained withhaematoxylin and show a qualitative reduction in smooth muscle actin anddesmin.

FIG. 5 shows immunoperoxidase labelling of fresh (A and C) anddecellularised (B and D) porcine bladder. Sections labelled withantibodies against collagen type IV (A and B) and laminin (C and D) werecounterstained with haematoxylin.

FIG. 6 shows immunofluorescence labelling of SM cells labelled with anantibody to smooth muscle actin (A). Hoescht 33258 dye was used toidentify all nuclei in an equivalent field of view (B). Scale bar 50 μm.

FIG. 7 (A) shows SM cell proliferation assessed by MTT assay over 7 daysand FIG. 7 (B) on day 7. The graphs show that in the presence of 10%(v/v) serum, proliferation of SM cells was not inhibited by mediumpreconditioned by incubation with the decellularised matrix. Absorbancewas measured in replicates of six and the calculated SEM plotted as xand y error bars.

FIG. 8 shows a phase-contrast micrograph showing porcine SM cellsgrowing up to the decellularised matrix (DM) indicating that the matrixwas not cytotoxic. Scale bar 50 μm.

FIG. 9 shows an H&E stained section showing porcine SM cells seeded ontothe decellularised porcine bladder matrix. (A) After 14 days in culture,there is a single layer of cells lining the surface of the matrix andsome cells are seen to have infiltrated. (B) Further matrix penetrationhas occurred after 21 days. The matrix showed no signs of degradation,Scale bar 100 μm.

FIG. 10 shows the phases of biological tissue stress-strain behaviour.

FIG. 11 shows biomechanical testing of fresh and decellularised porcinebladder tissue (n=6). Each test was performed in two directions:apex-to-base and transverse; and on samples from 5 different anatomicalregions of the bladder. Results are presented as mean values (+/−95%CI).

FIG. 12 shows determination of the (A) suture retention strength and (B)burst pressure of fresh and decellularised porcine bladder tissue.Suture retention tests were performed on bladder strips and burstpressure tests on intact bladder specimens. In each case, n=6 and theresults are presented as mean values (+/−95% CI).

FIG. 13 shows a burst pressure rig for testing of fresh anddecellularised bladder burst testing.

FIG. 14 is a bar chart comparison representation of suture retentionstrength between fresh bladder, decellularised bladder, freshpericardium, decellularised pericardium and SIS™ and PERMACOL™ implantsrespectively.

FIG. 15 is a bar chart comparison representation of mean elastin phaseslope between fresh bladder, decellularised bladder, fresh pericardium,decellularised pericardium and SIS™ and PERMACOL™ implants respectively.

FIG. 16 is a bar chart comparison representation of mean transitionstress between fresh bladder, decellularised bladder, fresh pericardium,decellularised pericardium and SIS™ and PERMACOL™ implants respectively.

FIG. 17 is a bar chart comparison representation of mean transitionstrain between fresh bladder, decellularised bladder, fresh pericardium,decellularised pericardium and SIS™ and PERMACOL™ implants respectively.

FIG. 18 is a bar chart comparison representation of mean thicknessbetween fresh bladder, decellularised bladder, fresh pericardium,decellularised pericardium and SIS™ and PERMACOL™ implants respectively.

FIG. 19 is a bar chart comparison representation of mean collagen slopebetween fresh bladder, decellularised bladder, fresh pericardium,decellularised pericardium and SIS™ and PERMACOL™ implants respectively.

FIG. 20 is a bar chart comparison representation of mean ultimatetensile strength between fresh bladder, decellularised bladder, freshpericardium, decellularised pericardium and SIS™ and PERMACOL™ implantsrespectively.

FIG. 21 is a bar chart comparison representation of mean failurestrength between fresh bladder, decellularised bladder, freshpericardium, decellularised pericardium and SIS™ and PERMACOL™ implantsrespectively.

FIG. 22 shows explanted fresh bladder tissue stained with (a)haematoxylin and eosin (b) Anti-CD3 (pan T-cell) and (c) Anti f4/80(macrophage). Magnification is at ×40 and arrows indicate implantedtissue.

FIG. 23 shows explanted acellular bladder biomaterial stained with (a)haematoxylin and eosin (b) Anti-CD3 (pan T-cell) and (c) Anti f4/80(macrophage). Magnification is at ×40 and arrows indicate implantedbiomaterials.

FIG. 24 shows a bar chart comparison representation of mean elastinphase slope between fresh decellularised bladder and decellularisedbladder that has been stored for 6, 9 and 30 months respectively.

FIG. 25 shows a bar chart comparison representation of mean collagenphase slope between fresh decellularised bladder and decellularisedbladder that has been stored for 6, 9 and 30 months respectively.

FIG. 26 shows a bar chart comparison representation of mean transitionstress between fresh decellularised bladder and decellularised bladderthat has been stored for 6, 9 and 30 months respectively.

FIG. 27 shows a bar chart comparison representation of mean ultimatetensile strength between fresh decellularised bladder and decellularisedbladder that has been stored for 6, 9 and 30 months respectively.

FIG. 28 shows a bar chart comparison representation of mean transitionstrength between fresh decellularised bladder and decellularised bladderthat has been stored for 6, 9 and 30 months respectively.

FIG. 29 shows a bar chart comparison representation of mean failurestrength between fresh decellularised bladder and decellularised bladderthat has been stored for 6, 9 and 30 months respectively.

DETAILED DESCRIPTION Specimen Procurement

Whole porcine bladders were obtained from a local abattoir within 4 h ofslaughter and transported to the laboratory on ice in sterile transportmedium (Hanks' balanced salt solution (HBSS) containing 10 mM HEPES pH7.6 and 10 KIU/ml Aprotinin (Trasylol, Bayer, Berkshire, UK).

Porcine Bladder Decellularisation

Intact bladders were washed in phosphate buffered saline (PBS)containing 0.1% w/v ethylene diamine tetra-acetic acid (EDTA) andAprotinin (10 KIU/ml) to inhibit protease activity. Subsequenttreatments, unless otherwise stated, were all carried out with proteaseinhibition. At each stage the intact bladder was distended with up to500 ml buffer through a funnel inserted into the bladder neck, closedwith Nalgene™ forceps (VWR International Ltd, Poole, UK) and immersed inthe same solution. The bladders were decellularised by incubating thebladder for 24 h at 4° C. in hypotonic Tris buffer (10 mM Tris, pH 8.0)followed by distension and incubation with agitation on an orbitalshaker for 24 h in 0.1% (w/v) sodium dodecyl sulphate (SDS) in hypotonicTris buffer at room temperature. Bladders were washed in PBS withoutprotease inhibition, before being incubated for 24 h in 50 U·ml⁻¹deoxyribonuclease I (Sigma, Poole, UK) and 1 Uml⁻¹ ribonuclease A(Sigma, Poole, UK) in 10 mM Tris-HCl pH 7.5 with gentle agitation at 37°C. Bladders were sterilized by incubation in 0.1% (v/v) peracetic acidin PBS for 3 h and finally, were washed in sterile PBS once for 24 hfollowed by 3 periods of 1 h under aseptic conditions. The resultingmaterial was stored in PBS at 4° C. for at least 6 months with no changein appearance or handling.

Histology and Microscopy

Fresh and decellularised tissue samples were fixed in 10% (v/v) neutralbuffered formalin, dehydrated and embedded in paraffin wax. Haematoxylinand eosin staining was used to evaluate the cellular content and generalhistoarchitecture of the porcine bladders. Miller's elastin staining wasused to evaluate the elastin content and the Van Gieson technique wasused for the identification of collagen I fibres (Bancroft and Stevens,Theory and Practise of Histological Techniques. London, ChurchillLivingstone, 1990).

Immuno-labelling of specific proteins was performed using an indirectimmunoperoxidase method as previously described (Booth et al,Labinvestigation 76, 843, 1997). Tris-buffered saline [(TBS), 0.05MTris-HCl, 0.15M NaCl, pH 7.6] was used as the diluent and wash bufferthroughout. Non-specific background staining was eliminated by blockingwith 10% (v/v) rabbit serum. Monoclonal antibodies were obtained againstcollagen type I (COL 1), smooth muscle actin (1A4), laminin (LAM89)(Sigma, Poole, UK), collagen type IV (cIV22), vimentin (V9) and desmin(D33) (Dako, High Wycombe, UK). Sections were incubated sequentially inprimary antibody for 1 h, biotinylated rabbit ant-mouse Ig (F(ab′)2fragments (Dako, High Wycombe, UK) for 30 min and strepavidin/HRP ABCcomplex (Dako, High Wycombe, UK) for 30 min, with washing between eachstep. Bound antibody was visualized using a 3,3′-diaminobenzidenesubstrate (DAB) reaction catalysed by H₂O₂. Sections were counterstainedwith haematoxylin, before being dehydrated, cleared and mounted in DPX(Sigma, Poole, UK). Omission of the primary antibody from the labellingprotocol and the use of irrelevant primary antibodies served as negativecontrols.

Biochemical Analysis

Three porcine bladders were decellularised for biochemical analysis andcomparison with six fresh, untreated porcine bladders. Unless otherwisestated, test solutions for analysis were prepared from samples of freshand decellularised matrix that had been freeze-dried to constant weight,hydrolysed by incubation with 6M HCl for 4 hours at 120° C. andneutralised to pH 7 with NaOH.

Glycosaminoglycan Assay

The amount of sulphated sugars (GAGs) was determined bydimethylmethylene blue binding (Enobakhare et al, Anal. Biochem. 243,189, 1996; Farndale et al, Biochim. Biophys, Acta., 883, 173, 1986).Briefly, test solutions were incubated with the dimethylmethylene bluesolution and the absorbance read at 525 nm. The amount of GAGs wascalculated by interpolation from a standard curve prepared usingchondroitin sulphate and phosphate assay buffer (0.1M sodium di-hydrogenorthophosphate, 0.1M di-sodium hydrogen orthophosphate, pH6.8) over arange of concentrations.

Hydroxyproline Assay

The amount of hydroxyproline was determined using a method based on thatdescribed elsewhere (Brown et al, Biotechniques 30, 38, 2001; Edwards etal, Clin Chem Acta 104, 161, 1980; Stegemann and Stadler Clin Chem Acta,18, 267, 1967). A range of hydroxyproline standards were prepared usingtrans-4-hydroxy-L-proline in hydroxyproline assay buffer [0.17M citricacid, 0.8% (v/v) acetic acid, 0.6M sodium acetate, 0.57M sodiumhydroxide and 20% (v/v) propan-1-ol pH6]. 50 μl of each standard andtest solution was aliquoted into a clear flat bottomed 96 well plate.Oxidation was achieved by adding 100 μl of chloramine T solution to eachwell. The plate was gently shaken for five minutes and 100 μl ofEhrlich's reagent added. The plate was covered and incubated at 60° C.for 45 minutes, before reading the absorbance at 570 nm. A standardcurve of hydroxyproline concentrations was plotted using the standardsolutions and the amount of hydroxyproline present in the test samplesdetermined. To measure the amount of denatured hydroxyproline, fresh anddecellularised tissue samples that had been freeze-dried to constantweight were digested with α-chymotrypsin prior to analysis (Bank et al,Matrix Biol. 16, 233, 1997).

DNA Assay

Fresh and decellularised tissue samples that had been freeze-dried toconstant weight were digested in papain buffer at 60° C. for 24 hours aspreviously described (Kim et al Anal Biochem, 174, 168, 1988; Labarca etal Anal Biochem, 102, 344 1980). Test solutions were incubated withHoechst 33258 dye solution and using a fluorometer, the plate was readusing excitation at 365 nm and emission at 458 nm. The amount of DNA wascalculated by interpolation from a standard curve prepared using calfthymus DNA solubilised in Tris buffered saline pH 7.6 over a range ofconcentrations.

Example 1

The bladder wall is composed primarily of collagen, elastin, and smoothmuscle and is organised in two major layers: the lamina propria and thedetrusor. The lamina propria consists of the urothelium, which lines theluminal surface, and an underlying connective tissue matrix thatcontains a dense layer of randomly oriented collagen fibres in which thecapillary network of the bladder is embedded. The majority of the laminapropria is constituted by a thick layer of collagen that functions tomaintain the shape of the bladder wall and to limit its overallcompliance (ratio of maximum volume divided by pressure). The detrusormuscle layer provides the contraction during voiding, and is composed ofmuscle fibres of 50 to 150 μm in diameter, and 20-50 μm apart andinterconnected with collagen bundles. Histological analysis of thedecellularised matrix showed that whilst the urothelium and smoothmuscle cells had been removed, the underlying histoarchitecture wasretained. (See FIGS. 2 and 3 and Example 6)

Example 2

Glycosaminoglycans (GAGs) are the main component of the ground substancein which cells, collagen (comprised of hydroxyproline, proline andglycine) and elastin fibres are embedded. Compared to fresh bladdertissue, the proportion of hydroxyproline and GAGs in decellularisedtissue samples relative to total dry weight, was significantly higherdue to the loss of other soluble proteins and cell components (Table 1).Table 1 below shows the biochemical characterisation of fresh anddecellularised porcine bladder tissue.

TABLE 1 Amount (μg/mg dry weight) Component Fresh bladder tissueDecellularised bladder tissue Total protein 345 (+/−20.4) 133.3(+/−7.6)* DNA 2.8 (+/−0.1) 0.1 (+/−0.1)* Hydroxyproline 46.8 (+/−2.0)82.0 (+/−4.3)* Denatured 2.0 (+/−0.1) 0.7 (+/−0.1)* HydroxyprolineGlycosaminoglycans 20.9 (+/−1.7) 53.2 (+/−3.1)* Results are presented asmean values (+/−95% CI) *indicates significant difference (student'st-test, p < 0.05)

Example 3

Tissue strips were dissected from the wall of fresh (within 24 h ofslaughter) and decellularised bladders using a scalpel and subjected tolow strain-rate uniaxial tensile loading to failure. In order to studypotential regional differences in the biomechanics of the bladder wall,five anatomical regions were tested, including the dorsal, ventral,lateral, trigone and lower body regions of the wall (FIG. 1). In eachregion, the anisotropy of the bladder wall was investigated by testingspecimens along the apex-to-base and transverse directions. For eachcase, tissue strips measuring 20×5 mm were dissected and mounted onto apurpose built titanium holder (Korossis et al J Heart Valve Dis 11, 463,2002). The holder was supported by a removable aluminium bracket thatallowed alignment of the two holder parts, defined the gauge length ofthe specimens and ensured that no load was imposed on the specimen untilthe start of the test. The gauge length of the specimens was defined bya 10 mm wide central block separating the two holder parts and screwedonto the bracket. Prior to clamping, the thickness of the specimens wasmeasured at 6 points along the long axis using a Mitutoyo thicknessgauge (Mitutoyo, Andover, UK) with a resolution of 0.01 mm and theaverage thickness was recorded. During clamping, care was taken to mountthe specimens under zero strain. Specifically, the specimen was floatedonto the smooth clamp surface with minimum handling and secured in itscompletely relaxed state. Once the specimen was clamped onto the holder,the holder with the supporting bracket was secured to a Howden tensilemachine and the bracket was removed. Testing was conducted inphysiological saline at 20° C.

Prior to loading to failure, the specimens were preconditioned by cyclicloading using a double-ramp wave function until a repeatableload-elongation response was observed. For all specimens tested, apreconditioning period of 50 cycles was sufficient to produce asteady-state response. Following preconditioning, the specimens wereloaded to failure using a positive ramp function at a rate of 10 mm/min.In order to obtain an accurate measure of the tissue gauge length, thetensile machine was set to produce a specimen preloading of 0.02 N,before the operating program started to acquire any data. Therefore,zero extension was taken at the point where a 0.02 N load was detected.The final gauge length of the specimen was calculated as the initialgauge length (10 mm) plus the extension that was needed to produce thespecified preloading. Failure was taken to occur when the first decreasein load was detected during extension. The mode of failure observed wasmiddle section necking and rupture for 90% of the specimens, independentof the specimen preparation, while the rest failed at the clampingpoint. During testing, load data from the load cell and extension datafrom the stroke of the tensile machine was acquired at a rate of 20 Hz.From the recorded load data the engineering stress (σ) was calculatedas:

$\sigma = \frac{F}{A_{o}}$where F is the acquired force in Newtons and A_(o) the originalcross-sectional area (CSA) of the undeformed specimen in mm². The CSAwas calculated as A_(o)=w×t, where w is the width of the tissue strip (5mm) and t its average thickness. The changes in thickness and widthduring preloading were considered negligible and were not taken intoaccount. The engineering strain (ε) was calculated from the extensiondata according to the formula:

$ɛ = {\frac{\Delta\; l}{l_{o}} \times 100}$where Δl is the extension of the specimen and l_(o) its final gaugelength.

The calculated stress-strain curves obtained for the specimens of eachgroup were averaged over the number of specimens in each group (n=6)using a mathematical analysis software package (Origin v6.0, Microbal).The stress-strain behavior for each specimen was analyzed by means ofsix parameters. These have been described elsewhere (Korossis et al JHeart Dis 11, 463, 2002) and included the elastin (El-E) and collagen(Col-E) phase slopes, transition stress (σ_(trans)) and strain(ε_(trans)), ultimate tensile strength (UTS) and failure strain(ε_(UTS)). The biomechanical parameters of the specimens in each testgroup were averaged, and compared by student t-test.

Example 4

Whole porcine bladders were obtained from a local abattoir within 4 h ofslaughter and transported to the laboratory on ice in sterile transportmedium. The bladder stromal tissue was stripped of urothelium (Southgateet al Lab Invest, 71, 583, 1994) and smooth muscle cells isolated aspreviously described for human smooth muscle cells (Kimuli et al BJU Int94, 859, 2004) were cultured in Dulbecco's Modified Eagle's Medium(DMEM) (Gibco, Paisley, UK) supplemented with 10% (v/v) fetal bovineserum (FBS) (Harlan, Loughborough, UK) and 1% (v/v) L-Glutamine (Sigma,Poole, UK) at 37° C. in a humidified atmosphere of 10% CO₂ in air.Morphological examination and immunohistological staining for smoothmuscle actin (1A4) (Sigma, Poole, UK) was used to confirm cell strainidentity and homogeneity of the cultures. Porcine smooth muscle (PSM)cell cultures were subcultured at confluence and maintained as finitecell lines through at least 15 passages.

Example 5

Decellularised bladder tissue was attached to the centre of a well in a6-well culture plate using sterile adhesive Steri-strips (3M,Manchester, UK). Porcine smooth muscle cells (SM) (passage 2 to 7) wereseeded into each well at a density of 1×10⁴ cells/ml. As negativecontrols, SM cells were seeded into wells containing steri-stripswithout matrix and into wells without steri-strips or matrix. Plateswere incubated at 37° C., 10% CO₂ for 48 h, 4 days or 12 days. Mediumwas then removed from each well, and the wells washed with PBS andstained/fixed with 1% (w/v) crystal violet (Sigma, Poole, UK) in 20%(v/v) ethanol before visualization by light microscopy.

Example 6

SM cells were suspended in growth medium at 1×10⁴ cells/ml and 200 μLwas added to the individual wells of a 96-well plate. Cells were left toattach at 37° C., 10% CO₂ for 2 h. A 5 cm² sheet of decellularisedbladder matrix was diced and added to 50 ml DMEM. After 24 h on a shakerthe medium was removed, filter-sterilised through a 0.2-μm filter andsupplemented with 0%, 5% or 10% (v/v) FBS. As a control, non-conditionedmedium was prepared in a similar way, except for omitting the dicedbladder matrix. The appropriate conditioned or non-conditioned mediumwas used to replace the medium on the cells in the 96-well plate inreplicates of six and the plates were incubated at 37° C. in ahumidified atmosphere of 10% CO₂ in air.3-[4,5-dimethyl(thiazol-2-yl)-3,5-diphery]tetrazolium bromide [(MTT)Sigma, Poole, UK] assays were used to compare the viability of SM cellsgrown in control or decellularised matrix-conditioned media. A singleplate was removed on days 0, 1, 4 and 7 to assess cell viability withthe MTT assay. MTT (200 μL, 0.5 mg/ml) was added to each well onappropriate days and left to incubate for 4 h at 37° C. The MTT wasreplaced by 200 μL of DMSO and mixed well to dissolve formazan crystals.The absorbance was read at 570 nm using a plate reader.

Example 7

In order to determine whether SM cells were able to repopulate thedecellularised tissue, suspensions of 2×10⁵ SM cells (at passages 2-7)in 200 μl DMEM were added to decellularised tissue samples in 6-wellplates and allowed to attach for 2 h after which time the wells wereflooded with complete DMEM. As a control, 200 μl of cell-free DMEM wasadded to tissue samples in wells that were flooded with complete DMEM asbefore. Seeded and non-seeded samples were collected on days 1, 3, 7, 14and 21 in duplicate, washed in PBS and fixed in 10% formalin forhistological assessment.

Example 8

The complete decellularisation of porcine bladder was confirmedhistologically. Compared to native bladder, matrices were completelydevoid of urothelium and there were no cells present within theunderlying tissue. This was confirmed by Hoechst 33258 staining ofsections to visualize cell nuclei (FIG. 2). Both Miller's elastin stainand the Van Gieson technique showed the general structure of thedecellularised matrix to resemble that of native bladder (FIG. 3).Immunolabelling with antibodies to αSMA, desmin and vimentin indicatedthat some poorly soluble cytoskeletal components of smooth muscle werenot removed by the decellularisation process (FIG. 4). Negative stainingfor collagen type IV and laminin, however, confirmed removal of thebasement membrane from the bladder lumen (FIG. 5).

Example 9

MTT assay showed that porcine cells, smooth muscle in origin (FIG. 6),underwent growth in a concentration-dependent manner as the percentageof serum in the medium increased from 0 to 10% (v/v) (FIG. 7). Smoothmuscle cells cultured in DMEM conditioned with the decellularised matrixshowed a similar concentration-dependent growth. There was nostatistical difference between the ultimate biomass of cells grown incontrol or conditioned medium under standard culture conditions.

Smooth muscle cells grew up to the decellularised matrix with noevidence of contact inhibition (FIG. 8) providing evidence that thedecellularised matrix was not cytotoxic. Smooth muscle cells were ableto attach and form a confluent monolayer of cells across the surface ofthe decellularised matrix after 3 days in culture. Under static cultureconditions, there was no cell penetration into the matrix after 7 days.After 14 days, however, cells had begun to infiltrate the matrix and by21 days had infiltrated to a depth approximately ⅓^(rd) that of thematrix (FIG. 9).

Example 10

The amount of DNA per mg dry weight of porcine bladder tissue before andafter decellularisation was 2.8 (+/−0.1) μg·mg⁻¹ and 0.1 (+/−0.1)μg·mg⁻¹, respectively (Table 1—see under Example 1). There was asignificant decrease in the DNA content of tissue afterdecellularisation (t-test; p<0.001). The concentrations ofhydroxyproline and GAGs per mg dry weight of porcine bladder tissuebefore and after decellularisation were also significantly different,with the relative proportion of each being significantly higher in thedecellularised tissue (t-test; p<0.001) reflecting the differentialremoval of other components.

Example 11

Tissue strips dissected from the wall of fresh and decellularisedbladders were subjected to low strain-rate uniaxial tensile loading tofailure. The stress-strain behaviour is shown in FIG. 10. In order tostudy potential regional differences in the biomechanics of the bladderwall, five anatomical regions were tested, including the dorsal,ventral, lateral, trigone and lower body regions of the wall. In eachregion, the anisotropy of the bladder wall was investigated by testingspecimens along the apex-to-base and transverse directions. Table 2shows the results of biomechanical tests, thickness, elastin andcollagen phase slope, transition stress and strain, ultimate tensilestrength and failure strain.

TABLE 2 Uniaxial tensile strength testing of fresh and decellularisedporcine bladder tissue. Fresh bladder tissue Decellularised bladdertissue Apex to Base (top row) Apex to Base (top row) Transverse (bottomrow) Transverse (bottom row) Bio-mechanical test Lower Lower results(Mean values) Dorsal Ventral Body Trigone Lateral Dorsal Ventral BodyTrigone Lateral Thickness (mm) 1.311 1.285 1.790 1.491 1.264 0.594 0.6960.647 0.829 0.604 1.299 1.222 1.577 1.309 1.425 0.552 0.614 0.589 0.8360.729 Elastin phase slope 2.87 3.39 2.18 3.21 3.81 37.8 6.09 16.0 10.321.9 [GPa (×10⁻⁵)] 3.78 1.91 1.85 3.34 3.87 33.4 15.2 9.48 10.1 10.8Collagen phase slope 9.41 6.51 21.4 20.6 15.2 44.2 36.4 37.2 49.2 78.5[GPa (×10⁻⁴)] 5.43 5.64 6.17 8.83 6.12 33.1 44.1 24.1 14.0 17.2Transition stress (MPa) 0.148 0.184 0.302 0.183 0.288 0.282 0.275 0.2700.522 0.496 0.110 0.166 0.141 0.151 0.192 0.156 0.322 0.197 0.164 0.175Transition strain (%) 126.52 119.17 153.63 87.11 152.72 27.63 37.8240.77 62.83 55.23 117.05 172.30 171.40 128.55 166.84 25.41 32.65 47.4251.90 33.21 Ultimate tensile 1.036 0.943 1.541 1.091 1.562 1.995 1.2502.018 2.254 3.058 strength (MPa) 0.741 0.709 0.907 0.815 0.833 1.1991.766 1.173 1.020 0.801 Failure strain (%) 266.63 314.73 237.20 158.65281.92 84.30 86.59 100.03 120.96 115.93 304.38 321.47 340.97 245.95316.73 80.59 79.69 110.14 139.60 85.67

The results showed that decellularised bladder tissue samples aresignificantly thinner than the fresh bladder samples. In fresh bladdersamples, the collagen phase slope values are higher in samples retrievedin an apex to base direction than in transverse samples. In addition,samples retrieved in an apex to base direction also have increasedultimate tensile strength compared to equivalent transverse samples andtheir failure strain is reduced.

As regards the collagen and elastin phase slope values of decellularisedbladder tissue samples, these are increased compared to fresh bladdertissue samples. Collected in an apex to base direction, decellularisedsamples have increased ultimate tensile strength compared to equivalentfresh samples. No significant differences in the ultimate tensilestrength of decellularised and fresh samples collected in a transversedirection were observed.

The results also showed that decellularised bladder samples havedecreased failure strain values as compared to fresh bladder samples andthat whilst transitional stress values do not differ significantlybetween fresh and decellularised bladder tissue samples, there is adecrease in the transitional strain values of decellularised samples.

Decreased strain values and increased collagen and elastin phase slopesindicate that the decellularised tissue is stiffer than fresh tissue(that the application of load results in less deformation per unitlength of tissue).

Example 12

The biomechanical properties of fresh (FIGS. 11A, C and E) anddecellularised (FIGS. 11B, D and E) bladder tissues were established byuniaxial tensile loading to failure of bladder wall strips from fiveanatomical regions of the bladder, namely the trigone, lower body,lateral, ventral and dorsal regions. Comparison of the results from thefive anatomical bladder regions showed that different regions of thebladder demonstrated different mechanical behaviour as depicted byvarying values for the collagen phase slope (FIGS. 11E and F), failurestrain (FIGS. 11A and B) and ultimate tensile strength (FIGS. 11C andD). Moreover, significant anisotropy was also found between theapex-to-base and transverse directions.

Collagen phase slope and average failure strain values weresignificantly changed following decellularisation, representingdecreased extensibility (t-test, p=<0.05), However, the ultimate tensilestrength (UTS) of the decellularised bladder wall was not significantlydifferent from that of the fresh bladder wall (t-test, p=>0.05).

Example 13

Bladder wall specimens were subjected to suture retention testing.Tissue strips measuring 10×5 mm were dissected from the lateral regionof the wall of fresh and decellularised bladders, along the apex-to-basedirection, and mounted onto the titanium holder, described above. Thespecimens were mounted so that only one end of the specimen was clampedto the holder. One polypropylene suture (4-0, Ethicon) with 2 mm bitedepth was attached to the other end of the specimen. The suture was thensecured to the upper part of the holder. Subsequently, the holder withthe supporting bracket was secured to the Howden tensile machine and thebracket was removed. The suture was then pulled under uniaxial loadingat a rate of 10 mm/min, and the suture pull-out force was recorded.

There was no significant difference in the ability of fresh anddecellularised bladder tissue to retain sutures under force (t-test,p>0.05); nor in the amount of pressure required to burst intact fresh ordecellularised bladders (t-test, p>0.05, FIG. 12).

Fresh and decellularised whole bladders were subjected to burst testing.For this purpose a burst pressure rig was developed (FIG. 13). The rigcomprised of a pressure vessel, which generated the test pressures, apressure gauge for measuring the applied hydrostatic pressures, and acontainer filled with saline, which accommodated the bladder undertesting. Pressurised air, supplied at a rate of 20 ml/s, was used topressurise the vessel, which was ¾s filled with saline. The inflow ofair in the vessel caused the pressurisation of the saline and thesubsequent filling and inflation of the bladder, which was connected viasilicone tubing to the pressure vessel. The pressure of the saline wasincreased until bursting of the bladder was achieved, and the maximumpressure just before bursting was recorded. There was no significantdifference in the burst pressure of fresh and decellularised porcinebladder tissue (FIG. 12).

Example 14

The biomechanical properties of fresh and decellularised bladder tissueswere compared with other membranous tissues that might be used in therepair of the bladder. These included fresh porcine pericardium,decellularised porcine pericardium, SIS™ and Permacol™ (FIGS. 14-21).

The decellularised porcine bladder, fresh and decellularised porcinepericardia all showed similar suture retention strength which wassignificantly lower (p<0.05; ANOVA) than that of SIS™ and Permacol™(FIG. 14). The mean elastin phase slope, collagen phase slope,transition stress and ultimate tensile strength of the decellularisedbladder material were not significantly different to those properties ofthe fresh bladder (p<0.05; ANOVA) compared to other biomaterials. Thesecomparisons indicate that decellularised bladder matrix would make themost suitable replacement for bladder tissue with regard tobiomechanical properties.

Example 15

In order to determine the in vivo biocompatibility of the acellularporcine bladder biomaterial, the reaction to the material was comparedto that of fresh porcine bladder tissue in a mouse subcutaneous implantmodel.

Following a short term general anaesthesia, two 5 mm² pieces of fresh oracellular porcine bladder were implanted subcutaneously in normal mice(female 6-8 week old mf-1 hairless mice; n=3 in each group). Mice weresacrificed at three months and the implants and the overlying skin wereretrieved. The explanted tissues were cryoembedded in OCT and sections(5 □m) cut in a cryostat. Representative sections from throughout thetissue were stained with haematoxylin and eosin to visualize the generalhistioarchitecture. The cellular infiltrate in the tissues wasdetermined by immunoperoxidase staining using rat monoclonal antibodiesto mouse CD3 (pan T-cells; IgG2a; Caltag) and F4/80 (macrophages; IgG2b;Caltag). Goat anti-rat IgG conjugated to biotin was used as thesecondary antibody. Appropriate negative controls were included.Representative images were captured digitally.

The fresh tissue explants were encapsulated by cells which werepredominantly F4/80 (macrophage) or C3 (T-cell) positive (FIG. 22). Thetissues showed signs of vacuolation/disintegration and there were veryfew cells present within the matrix of the implanted fresh tissues. Thepattern was indicative of a foreign body response to the implanted freshtissues.

The acellular porcine bladder biomaterial explants showed minimalencapsulation and the biomaterial appeared to be integrated into themouse skin (FIG. 23). There were sparse CD3 positive T-cells around orwithin the biomaterial, although CD3 positive cells were clearly visiblein the epidermis of the mouse skin (normal). There were signs ofcellular infiltration into the matrix of the biomaterial, with some ofthese cells being F4/80 positive. Other cells had a fibroblasticappearance. The pattern was indicative of a wound healing-type reactionas judged by the presence of dispersed F4/80 positive cells in theabsence of T-cells.

In contrast to the fresh bladder tissue which showed an overt foreignbody response when implanted subcutaneously in mice, the acellularporcine bladder biomaterial showed good integration into the mouse skinindicating that the material was biocompatible in this model. Thisexample provides evidence for the use of acellular porcine bladderbiomaterials as appropriate material for wound healing.

Example 16

Aged stored samples of 6, 9, and 30 months old were tested for a varietyof biomechanical strength parameters as hereinbefore described andcompared to freshly (24 hour old) prepared decellularised bladdermaterial. With references to FIGS. 24 to 29, there is presented barchart results for mean elastin phase slope, collagen phase slope,transition stress, ultimate tensile strength, transition strain andfailure strain respectively.

The results (Table 3) from uniaxial tensile testing indicated that therewas no significant differences in any of the biomechanical parametersstudied between decellularised scaffolds tested 24 hours post treatmentand decellularised scaffolds tested 6, 9 and 30 months post treatment.P-values were calculated and obtained by ANOVA (Table 4).

These results indicate that the mechanical integrity of thedecellularised bladder scaffolds remains intact for periods up to atleast 30 months. In other words the shelf life of the material is atleast 30 months.

TABLE 3 SELF LIFE STUDY DECELLULARISED BLADDER 0 MONTHS (Lateral, Apexto Base) Test E CoII-E σ_(T) ε_(T) σ_(UTS) ε_(UTS) Thick Width Length No(GPa) (GPa) (MPa) (%) (MPa) (%) (mm) (mm) (mm) 69 7.91E−05 5.04E−030.473 78.53 3.871 161.40 0.590 5.000 13.320 80 4.80E−04 1.60E−02 0.73246.03 3.702 79.08 0.433 5.000 12.580 87 9.77E−05 2.51E−03 0.283 41.141.602 107.30 0.790 5.000 13.700 Mean 2.19E−04 7.85E−03 0.496 55.23 3.058115.93 0.604 StDev 1.85E−04 5.85E−03 0.184 16.59 1.032 34.16 0.146 Count3 3 3 3 3 3 3 95% C.I. 2.09E−04 6.63E−03 0.208 18.78 1.168 38.65 0.165Test E CoII-E σ_(T) ε_(T) σ_(UTS) ε_(UTS) Thick Width Length No (GPa)(GPa) (MPa) (%) (MPa) (%) (mm) (mm) (mm) DECELLULARISED BLADDER 6 MONTHS11  1.22E−04 3.74E−03 0.184 23.54 1.405 62.28 0.743 5.000 15.780 12 4.71E−05 4.31E−03 0.233 36.18 1.626 78.66 0.741 5.000 16.590 13 3.06E−04 5.24E−03 0.391 45.30 1.856 95.35 0.737 5.000 18.580 Mean1.58E−04 4.43E−03 0.269 35.01 1.629 78.76 0.740 StDev 1.09E−04 6.18E−040.088 8.92 0.184 13.50 0.002 Count 3 3 3 3 3 3 3 95% C.I. 1.23E−047.00E−04 0.100 10.10 0.208 15.28 0.003 DECELLULARISED BLADDER 9 MONTHS 81.22E−04 2.79E−03 0.142 43.18 1.334 97.20 0.862 5.000 15.370 9 1.76E−042.26E−03 0.248 67.82 1.299 139.50 0.870 5.000 13.240 10  2.76E−044.54E−03 0.425 65.44 2.088 122.10 0.874 5.000 14.470 Mean 1.91E−043.20E−03 0.272 58.81 1.574 119.60 0.869 StDev 6.36E−05 9.74E−04 0.11711.10 0.364 17.36 0.005 Count 3 3 3 3 3 3 3 95% C.I. 7.20E−05 1.10E−030.132 12.56 0.412 19.64 0.006 DECELLULARISED BLADDER 30 months 59.44E−05 1.10E−02 0.407 38.81 2.75 55.28 0.491 5.000 16.540 6 1.59E−047.04E−03 0.272 40.09 2.06 75.34 0.496 5.000 13.950 7 1.01E−04 5.63E−030.320 47.50 1.88 91.39 0.491 5.000 16.800 Mean 1.18E−04 7.89E−03 0.33342.13 2.23 74.00 0.493 StDev 2.90E−05 2.27E−03 0.056 3.83 0.37 14.770.002 Count 3 3 3 3 3 3 3 95% C.I. 3.28E−05 2.57E−03 0.063 4.33 0.4216.72 0.003

TABLE 4 SELF LIFE ANOVA E (GPa) CoII-E (GPa) 0-MONTHS 6-MONTHS 9-MONTHS30-MONTHS 0-MONTHS 6-MONTHS 9-MONTHS 30-MONTHS 7.91E−05 1.22E−041.22E−04 9.44E−05 5.04E−03 3.74E−03 2.79E−03 1.10E−02 4.80E−04 4.71E−051.76E−04 1.59E−04 1.60E−02 4.31E−03 2.26E−03 7.04E−03 9.77E−05 3.06E−042.76E−04 1.01E−04 2.51E−03 5.24E−03 4.54E−03 5.63E−03 P-value 0.7100.850 0.488 P-value 0.457 0.330 0.993 σ_(T) (MPa) ε_(T) (%) 0-MONTHS6-MONTHS 9-MONTHS 30-MONTHS 0-MONTHS 6-MONTHS 9-MONTHS 30-MONTHS 0.4730.184 0.142 0.407 78.53 23.54 43.18 38.81 0.732 0.233 0.248 0.272 46.0336.18 67.82 40.09 0.283 0.391 0.425 0.320 41.14 45.30 65.44 47.50P-value 0.191 0.279 0.297 P-value 0.204 0.812 0.338 σ_(UTS) (MPa)ε_(UTS) (%) 0-MONTHS 6-MONTHS 9-MONTHS 30-MONTHS 0-MONTHS 6-MONTHS9-MONTHS 30-MONTHS 3.871 1.405 1.334 2.747 161.40 62.28 97.20 55.283.702 1.626 1.299 2.061 79.08 78.66 139.50 75.34 3.602 1.856 2.088 1.883107.30 95.35 122.10 91.39 P-value 0.126 0.127 0.346 P-value 0.226 0.8990.186

The invention claimed is:
 1. A method of implanting a tissue matrix inbladder reconstruction or replacement surgery comprising: immersing adistensible membranous sac in a buffer solution at a mild alkaline pHwhich includes active amounts of a proteolytic inhibitor; (ii)distending the distensible membranous sac by introducing a sufficientvolume of the same buffer solution into the interior cavity of the sac;(iii) continuing decellularisation of the sac by replacing andintroducing fresh solutions around the exterior surface of the sacand/or into the sac interior so as to maintain distension of the sacduring recellularisation; and (iv) implanting the whole decellulariseddistensible sac or a portion thereof into a donor.
 2. The method ofclaim 1 wherein steps (i) and (ii) are reversed.
 3. The method of claim1, wherein the distensible membranous sac is a whole bladder.
 4. Themethod of claim 1, wherein the distensible membranous sac is derivedfrom a pig or a human.
 5. A method of urological tissue engineeringreplacement comprising: (i) immersing a bladder material in a buffersolution at a mild alkaline pH which includes a proteolytic inhibitor;(ii) expanding the bladder by introducing a sufficient volume of thesame buffer solution into an interior cavity of the bladder so as tostretch and thin the bladder wall; and (iii) continuingdecellularisation of the bladder by replacing and introducing freshsolutions both around the exterior surface of the bladder and into thebladder interior itself so as to maintain expansion of the bladderduring decellularisation and to maintain the histoarchitecture of thebladder material; and (iv) implanting the bladder material or a portionthereof into a donor, wherein the method of tissue engineeringreplacement or tissue repair is selected from the group consisting ofcosmetic surgery and scaffold for tissue-engineering.